Bottom Line:
Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt.Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.

Background: Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.

Results: We addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.

Conclusions: Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.

Figure 1: Effects of tumor necrosis factor (TNF)-α and exogenous ceramide on L6 myotube characteristics. (A) Differentiated myotubes treated for 3 days with 15 ng/ml TNF-α or 5 μmol/l C6 ceramide were immunostained for sarcomeric myosin heavy chain (MHC), and their surfaces were evaluated by using Image J software. The graph shows the myotube area (mean ± SE) expressed as a percentage of the field surface, as determined in ten fields, and is representative of at least three experiments. Different from control: **P = 0.01; ***P = 0.001. (B) The MHC content of myotubes treated for 3 consecutive days with TNF-α or C6 ceramide at the indicated concentrations was evaluated by ELISA. The mean ± SE of three measurements expressed as a percentage of control values is shown. The mean control value was 75 ± 11 μg of MHC per 120,000 cells. *Different from control: P < 0.05; P ≤ 0.001. (C) The creatine kinase activity of myotubes treated for 3 days with 15 ng/ml TNF-α or 5 μmol/l C6 ceramide was assayed in triplicate (mean ± SE).*Different from control: P < 0.05.

Mentions:
In differentiated myotubes of the L6 cell line submitted to 15 ng/ml recombinant TNF-α treatment for 3 days, cell atrophy was present, as evidenced by a significant decrease in cell surface, as already reported [20] (Figure 1a). Other parameters reflecting the functional status of the differentiated muscle cells were also significantly reduced by TNF-α treatment, such as the myosin heavy chain (MHC) content, as evaluated by ELISA (Figure 1b), and the creatine kinase (CK) activity (Figure 1c). Similarly, TNF-α treatment induced a decrease in cell surface in myotubes derived from the C2C12 line (see Additional file 1). We verified that in these conditions TNF-α induced no change in cell viability (not shown). The effects of TNF-α on cellular levels of sphingolipids were assessed in L6 myotubes by tandem mass spectrometry (MS/MS). As expected, TNF-α treatment was able to increase the levels of ceramide, in this case, by 35%. The bulk of the increase mainly concerned a subset of ceramide molecular species: C16:0, C24:1, and C18:0 ceramides (Table 1). TNF-α action also resulted in a 30% decrease in sphingomyelin, especially the C16:0 and C24:1 molecular species, reflecting an activation of sphingomyelinases (Table 1). To assess whether ceramide accumulation could explain the atrophic effects of TNF-α, we investigated the effects of myotube treatment by exogenous ceramide. Interestingly, in both the L6 and C2C12 cell lines, the atrophic effects of TNF-α were mimicked by the addition to the culture medium of cell-permeating short-chain ceramides, particularly C6 ceramide (Figure 1a-c; see Additional file 1), suggesting that myotube atrophy might result from TNF-α-induced ceramide accumulation.

Figure 1: Effects of tumor necrosis factor (TNF)-α and exogenous ceramide on L6 myotube characteristics. (A) Differentiated myotubes treated for 3 days with 15 ng/ml TNF-α or 5 μmol/l C6 ceramide were immunostained for sarcomeric myosin heavy chain (MHC), and their surfaces were evaluated by using Image J software. The graph shows the myotube area (mean ± SE) expressed as a percentage of the field surface, as determined in ten fields, and is representative of at least three experiments. Different from control: **P = 0.01; ***P = 0.001. (B) The MHC content of myotubes treated for 3 consecutive days with TNF-α or C6 ceramide at the indicated concentrations was evaluated by ELISA. The mean ± SE of three measurements expressed as a percentage of control values is shown. The mean control value was 75 ± 11 μg of MHC per 120,000 cells. *Different from control: P < 0.05; P ≤ 0.001. (C) The creatine kinase activity of myotubes treated for 3 days with 15 ng/ml TNF-α or 5 μmol/l C6 ceramide was assayed in triplicate (mean ± SE).*Different from control: P < 0.05.

Mentions:
In differentiated myotubes of the L6 cell line submitted to 15 ng/ml recombinant TNF-α treatment for 3 days, cell atrophy was present, as evidenced by a significant decrease in cell surface, as already reported [20] (Figure 1a). Other parameters reflecting the functional status of the differentiated muscle cells were also significantly reduced by TNF-α treatment, such as the myosin heavy chain (MHC) content, as evaluated by ELISA (Figure 1b), and the creatine kinase (CK) activity (Figure 1c). Similarly, TNF-α treatment induced a decrease in cell surface in myotubes derived from the C2C12 line (see Additional file 1). We verified that in these conditions TNF-α induced no change in cell viability (not shown). The effects of TNF-α on cellular levels of sphingolipids were assessed in L6 myotubes by tandem mass spectrometry (MS/MS). As expected, TNF-α treatment was able to increase the levels of ceramide, in this case, by 35%. The bulk of the increase mainly concerned a subset of ceramide molecular species: C16:0, C24:1, and C18:0 ceramides (Table 1). TNF-α action also resulted in a 30% decrease in sphingomyelin, especially the C16:0 and C24:1 molecular species, reflecting an activation of sphingomyelinases (Table 1). To assess whether ceramide accumulation could explain the atrophic effects of TNF-α, we investigated the effects of myotube treatment by exogenous ceramide. Interestingly, in both the L6 and C2C12 cell lines, the atrophic effects of TNF-α were mimicked by the addition to the culture medium of cell-permeating short-chain ceramides, particularly C6 ceramide (Figure 1a-c; see Additional file 1), suggesting that myotube atrophy might result from TNF-α-induced ceramide accumulation.

Bottom Line:
Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt.Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.

Background: Muscle atrophy associated with various pathophysiological conditions represents a major health problem, because of its contribution to the deterioration of patient status and its effect on mortality. Although the involvement of pro-inflammatory cytokines in this process is well recognized, the role of sphingolipid metabolism alterations induced by the cytokines has received little attention.

Results: We addressed this question both in vitro using differentiated myotubes treated with TNF-α, and in vivo in a murine model of tumor-induced cachexia. Myotube atrophy induced by TNF-α was accompanied by a substantial increase in cell ceramide levels, and could be mimicked by the addition of exogenous ceramides. It could be prevented by the addition of ceramide-synthesis inhibitors that targeted either the de novo pathway (myriocin), or the sphingomyelinases (GW4869 and 3-O-methylsphingomyelin). In the presence of TNF-α, ceramide-synthesis inhibitors significantly increased protein synthesis and decreased proteolysis. In parallel, they lowered the expression of both the Atrogin-1 and LC3b genes, involved in muscle protein degradation by proteasome and in autophagic proteolysis, respectively, and increased the proportion of inactive, phosphorylated Foxo3 transcription factor. Furthermore, these inhibitors increased the expression and/or phosphorylation levels of key factors regulating protein metabolism, including phospholipase D, an activator of mammalian target of rapamycin (mTOR), and the mTOR substrates S6K1 and Akt. In vivo, C26 carcinoma implantation induced a substantial increase in muscle ceramide, together with drastic muscle atrophy. Treatment of the animals with myriocin reduced the expression of the atrogenes Foxo3 and Atrogin-1, and partially protected muscle tissue from atrophy.

Conclusions: Ceramide accumulation induced by TNF-α or tumor development participates in the mechanism of muscle-cell atrophy, and sphingolipid metabolism is a logical target for pharmacological or nutritional interventions aiming at preserving muscle mass in pathological situations.